As for Koji used in the production of alcoholic beverages, there are solid koji, which is cultured such that a spore of mold is inoculated into a raw material after treatment with cooking or the like and then cultivated, and a liquid koji, which is cultured such that a liquid-medium is prepared by adding a raw material and other nutrients to water, and then inoculated with spores of mold, mycelium cultured in advance or the like into the liquid medium, followed by cultivation.
In the conventional production of alcoholic beverages or fermented foods and drinks, such as sake, shochu, soy sauce, fermented soybean paste, and sweet sake, the so-called solid koji prepared by a solid culture method has been widely used. The solid culture method is a culture method by which a koji mold such as Aspergillus kawachii, Aspergillus awamori, Aspergillus niger, Aspergillus oryzae, or Aspergillus sojae is diffused on a solid raw material such as steam-cooked cereals to allow a koji mold to grow on the solid surface.
For instance, for the production of shochu, Aspergillus kawachii, Aspergillus awamori, and so on have been widely used. However, as the solid culture method is a culture system in which a raw material and the koji mold disperse unevenly, it is difficult to equalize factors such as temperature, water content, and various nutrients. Therefore, the culture can be very complicated to be controlled. In addition, the production of koji tends to be carried out in an open state. In this case, care should be exercised in quality control with respect to contamination with other bacteria. Therefore, it is unsuitable for a large-scale production.
In contrast, the liquid culture method is easily subjected to culture control and quality control, so that it is suitable for efficient production. However, there is a problem for example, in that a sufficient enzymatic activity required for brewing shochu is not obtained. Therefore, there are few examples in which a culture product obtained by the liquid culture of a koji mold was really used as a shochu koji. Here, the “culture product” obtained by the liquid culture method means a culture product itself obtained by the liquid culture methods (hereinafter, also referred to as a “liquid koji”) as well as a culture liquid, molds, a concentrate thereof, or a dried product thereof.
A major reason for no use of the culture product obtained by the liquid culture method in the production of fermented foods and drinks such as shochu etc., is that the behavior of a koji mold to produce an enzyme such as amylase or cellulase in the liquid culture is known to be much different from one in the solid culture, and the productivity thereof is also known to be decreased overall (see Non-Patent Document 1).
Typically, in the production of alcoholic beverages including shochu, alcohol is generated by parallel fermentation of two types. Therefore, saccharolytic enzymes from a koji mold, which affect on the supply of glucose to the koji mold, particularly glucoamylase (hereinafter sometimes abbreviated as GA) and acid-stable α-amylase (hereinafter sometimes abbreviated as ASAA) are key enzymes in alcoholic fermentation. However, it is known that the activity of glucoamylase in the culture product obtained by the liquid culture method is remarkably low and the production behavior thereof is also much different from one in the solid culture (see Non-Patent Document 2).
As a method of improving the glucoamylase activity of a koji mold, there are two methods which have been reported, one cultures the koji mold while giving a stress on the growth of mycelium (see Patent Document 1) and the other adds roasted cereals to a koji mold culture medium (see Patent Document 2). The method disclosed in Patent Document 1 carries out a culture on a porous membrane or in an inclusive fixing agent having aperture to express a novel gene glaB that encodes glucoamylase, to enhance the enzymatic activities. Thus, the method requires strict control or a specific culture device and thus it is not practical. In addition, the method disclosed in Patent Document 2 is a method of cultivating a koji mold in a liquid medium using the roasted cereals as, at least, a portion of the raw material. Accordingly, said method requires an additional production step of roasting cereals.
Therefore, the inventors of the present invention offered an invention related to a method of culturing a koji mold using a liquid medium that contains hardly decomposable saccharides for the koji mold (see Patent Document 3). According to this invention, the koji mold culture product having a high activity of glycolytic enzymes such as glucoamylase, which can be used in the production of alcoholic beverages or fermented foods and drinks, can be obtained conveniently and inexpensively.
On the other hand, recently, the molecular biological analysis on acid-stable α-amylase has been started (see Non-Patent Document 3). In this case, it is reported as follows: A white koji mold has two different amylase genes which are respectively responsible for two different characteristics, acid-unstable α-amylase and acid-stable α-amylase. However, the expression behaviors of the respective genes are much different from each other. In the liquid culture, the acid-unstable α-amylase can be produced in sufficient amount, while the acid-stable α-amylase, a key enzyme for brewing shochu, is hardly produced.
In the production of shochu, it is brewed under a low-pH environment for preventing a shochu mash from putrefaction. However, the acid-unstable α-amylase does not contribute to glycolysis in shochu brewing because the acid-unstable α-amylase is deactivated promptly under low-pH conditions. Therefore, for the production of shochu, it is indispensable to produce acid-stable α-amylase with high yield, which may contribute to the glycolysis in shochu brewing, by the liquid culture of a koji mold.
In the past, there was a report in which the behavior of a koji mold in a liquid culture to produce acid-stable α-amylase was investigated. The method uses a synthetic medium containing peptone and a citrate buffer solution and requires a cultivation time of 100 hours or more. Therefore, it is hard to say to be a process of manufacturing a liquid koji applicable to the actual shochu brewing (see Non-Patent Document 4).    Patent Document 1: JP-A 11-225746    Patent Document 2: JP-A 2001-321154    Patent Document 3: JP-A 2003-265165    Non-Patent Document 1: Iwashita K. et al: Biosci. Biotechnol. Biochem., 62, 1938-1946(1998), Yuichi Yamane et al: published by Brewing Society of Japan, 99, 84-92(2004)    Non-Patent Document 2: Hata Y. et al: J. Ferment. Bioeng., 84, 532-537(1997), Hata Y. et al: Gene, 207, 127-134(1998), Ishida H. et al: J. Ferment. Bioeng., 86, 301-307(1998), Ishida H. et al: Curr Genet., 37, 373.-379(2000)    Non-patent Document 3: Nagamine K. et al: Biosci. Biotechnol. Biochem., 67, 2194-2202(2003)    Non-Patent Document 4: Sudo S. et al: J. Ferment. Bioeng., 76, 105-110(1993), Sudo S. et al: J. Ferment. Bioeng., 77, 483-489(1994), Shigetoshi Sudo et al: published by Brewing Society of Japan, 89, 768-774(1994)